Estrogen protects the cardiovascular system from injury by complex mechanisms that have both short- and long-term components. Longer- term estrogen actions occurs following hours of estrogen exposure and is mediated by estrogen receptors acting in the nucleus as transcription factors for growth-related genes. The rapid effects of estrogen, by contrast, occurs in a seconds-to-minutes time frame and do not require gene transcription. Project 5 of the SCOR application addresses the molecular mechanisms underlying one of the most important of these rapid effects-vasodilation. The signaling pathways underlying this non- genomic action of estrogen are incompletely understood but new data support they involve the action of nitric oxide synthase in vascular cells, elevation of nitric oxide, stimulation of vascular smooth muscle cell soluble guanylyl cyclase, and substrate phosphorylation by cGMP- dependent protein kinase (PKG). Although the key substrate(s) for PKG is unknown, new data support one likely possibility is the smooth muscle large conductance Ca2+-activated K+ (BKCa) channel. Blockade of BKCa channels in vascular rings significantly reduces estrogen-induced vasodilation, demonstrating the central role that BKCa channels in vascular rings significantly reduces estrogen-induced vasodilation, demonstrating the central role that BKCa channels play in the acute physiological response to estrogen. Experiments in Aim 1 will use BKCa channels in vascular rings significantly reduces estrogen-induced vasodilation, demonstrating the central role that BKCa channels play in the acute physiological response to estrogen. Experiments in Aim 1 will use BKCa channel activity recorded from single smooth muscle cells dissociated from mouse aorta as an assay for characterizing upstream elements in the estrogen signaling pathway that targets BKCa channels. Experiments will assess the relative contributions of endothelial and smooth muscle cells, the types of estrogen receptor involved, and the requirement for nitric oxide synthase activation. Experiments in Aim 2 will focus on the molecular mechanism by which BKCa channel activity is regulated by estrogen. We will identify the molecular variants of BKCa channel activity is regulated by estrogen. We will identify the molecular variants. variants of BKCa channels that are expressed in smooth muscles cells from mouse aorta and study the recombinant channels expressed heterologously in order to test the hypothesis that estrogen brings about BKCa channel activation through a direct PKG-dependent phosphorylation of the channel protein. Such information is essential to further understand the protective effects of estrogen on vascular tissue and may form the basis for development of new therapies for cardiovascular disease.